苹果液泡膜蔗糖转运蛋白基因MdSUT4的表达分析与功能鉴定

以新疆红肉苹果杂种一代优系‘红脆1号’为试材,克隆MdSUT4并原核诱导获得其重组蛋白,对其进行生物信息学分析,测定其在不同组织及不同发育时期果实中的表达,通过亚细胞定位及转基因鉴定其功能。结果发现,MdSUT4编码的蛋白大小为55 kD左右,定位于8号染色体,由5个外显子和4个内含子组成;糖代谢相关基因系统进化树分析发现,MdSUT4与AtSUC4、PpSUT4在同一个进化支上;定量表达分析发现,MdSUT4在苹果的花、叶和幼果中均有较高的表达,且在果实发育中的表达水平与蔗糖含量显著负相关;MdSUT4启动子含有与糖信号及激素信号相关的顺式作用元件;在‘王林’苹果愈伤组织中过表达MdSUT4,能降低蔗糖含量,但却提高类黄酮含量;MdSUT4定位于‘王林’愈伤组织原生质体的液泡膜上。以上结果表明,MdSUT4可能参与了蔗糖从液泡膜中的外排,并可能促进类黄酮的合成。

Expression Analysis and Functional Identification of a Vacuolar Sucrose Transporter Gene MdSUT4 in Apple

MdSUT4 was cloned from'Hongcui 1'apple and the recombinant protein was obtained by the prokaryotic induction technology,then,it was analyzed using bio-information.The expression level of MdSUT4 in different tissues and different development stages were studied by the qRT-PCR,and we also identified the function of MdSUT4 by the subcellular localization and transgenosis.The results showed that the molecular mass of MdSUT4 was about 55 kD,it was located in the chromosome 8 of the apple genome,included 5 exons and 4 introns.A phylogenetic tree indicated MdSUT4,AtSUC4 and PpSUT4 were located in the same evolutionary branch.The qRT-PCR showed that the MdSUT4 has the higher expression level in flowers,leaves and young fruits of apples,and its expression had a significant negative correlation with the content of sucrose during the development stage of fruits.The promoter of MdSUT4 contained several typical cis-acting elements,including sugar signaling responsive elements and phytohormone responsive elements.We overexpressed MdSUT4 in'Orin'callus,then found that it could increase the content of flavonoid and decrease the content of sucrose.MdSUT4 was located in the tonoplast of'Orin'callus.These results indicated that MdSUT4 might participate in the effiux of sucrose from the vacuole and promote the synthesis of flavonoid.